J Periodontol • March 2011

Study of Orthophosphate,
Pyrophosphate, and Pyrophosphatase
in Saliva With Reference to Calculus
Formation and Inhibition
A.R. Pradeep,* Esha Agarwal,* Arjun Raju P.,† M.S. Narayana Rao,‡
and Mohamed Faizuddin§

Background: A large amount of calculus may hamper the

efficacy of daily oral hygiene and thereby accelerate plaque for-
mation. Salivary concentrations of orthophosphate and pyro-
phosphate are important in preventing calculus formation.
Activity of orthophosphate, pyrophosphate, and pyrophospha-
tase was studied in whole saliva in calculus-forming groups and
plaque-forming groups.

D
ental calculus occurs in most
Methods: The material for this study consists of 60 healthy adults worldwide.1 Studies2,3 have
individuals (age range: 15 to 30 years; mean age: 22 years). shown that the combination of
Depending on calculus index score, individuals were divided dental calculus, plaque, and age is stron-
into four groups, each of 15 patients: Group 1, calculus index gly correlated with the severity of peri-
score 0 to 0.6; Group 2, calculus index score 0.7 to 1.8; Group odontal disease. Previous studies4-6 have
3, calculus index score 1.9 to 3; and Group 4, plaque group indicated that a large amount of calculus
where index varied from 0 to 3. The saliva was collected and may hamper the efficacy of daily oral
biochemically analyzed for concentration of orthophosphate, hygiene and thereby accelerate plaque
pyrophosphate, and pyrophosphatase. formation, the accumulation of which in-
Results: The mean values of orthophosphate in Groups 1, 2, itiates the inflammatory reaction in the
3, and 4 were 0.2559, 1.3639, 1.7311, and 0.1868 mM, respec- gingiva that leads to periodontitis.7
tively. The mean values of pyrophosphate in Groups 1, 2, 3, and The phenomenon of calculus forma-
4 were 0.3258, 0.1091, 0.0314, and 0.3860 mM, respectively. tion and its inhibition has been widely dis-
The mean values of pyrophosphatase in Groups 1, 2, 3, and cussed in the literature. Such substances
4 were 10.7937, 15.4249, 27.2900, and 7.5427 units/ml, re- as orthophosphate and pyrophosphatase
spectively. find their importance in the formation of
Conclusions: A holistic approach toward the control of peri- calculus. Orthophosphate is directly re-
odontal disease should include antiplaque and anticalculus lated to its action by competing with pyro-
agents. The results are conclusive that the components ortho- phosphate8 and can even alter the effect
phosphate, pyrophosphate, and pyrophosphatase present of pyrophosphate on inhibition of calcifi-
in saliva have a very significant role to play in formation and cation and thus create an environment
inhibition of calculus. This study reinforces the idea of using highly suitable for calculus deposition,
pyrophosphate and newer bisphosphonates as potential anti- whereas enzyme pyrophosphatase in-
calculus agents. J Periodontol 2011;82:445-451. hibits the action of pyrophosphate and
helps in calculus formation indirectly by
KEY WORDS
converting pyrophosphate to orthophos-
Calculi; dental plaque; inorganic pyrophosphate; phosphate; phate.9,10 Enzyme alkaline phosphatase,9
saliva; statistics. present in saliva and in plaque, releases
inorganic orthophosphate from organic
* Department of Periodontics, Government Dental College and Research Institute, Bangalore, phosphate, increasing the concentration
Karnataka, India.
† Medical Student, Bangalore Medical College and Research Institute, Bangalore, Karnataka, of orthophosphate locally, which reacts
India. with calcium ions leading to precipita-
‡ Former Professor, Government Dental College and Research Institute, Bangalore,
Karnataka, India. tion of insoluble calcium apatite crystals.
§ Department of Periodontics, M.R. Ambedkar Dental College and Hospital, Cooke Town,
Bangalore, Karnataka, India.
doi: 10.1902/jop.2010.100355

445
Orthophosphate, Pyrophosphate, and Pyrophosphatase in Saliva
Pyrophosphate, a byproduct of many biosynthetic
reactions,10 present in saliva inhibits crystallization
and competes with orthophosphate8 for minerals,
thus having a strong inhibitory effect on plaque miner-
alization. Various studies in this regard have proved
that severity of calculus formation is inversely propor-
tional to pyrophosphate concentration in saliva.8,11-14
Apart from its activity in saliva, there have been var-
ious reports in the literature wherein pyrophosphate
plays an equally important role. Fleisch et al.15-17 re-
ported its role in inhibition of urinary calculus. Its role
in inhibition of calcification of cartilage and colla-
gen18-20 and its activity in enamel and dentin21 have
also been reported.
To the best of our knowledge no single study has
correlated the concentrations of orthophosphate, py-
rophosphate, and pyrophosphatase with calculus
index scores. On the basis of this background, the
activity of orthophosphate, pyrophosphate, and pyro-
phosphatase was studied in whole saliva in calculus-
forming groups and a plaque-forming group.
MATERIALS AND METHODS
The material for this study consisted of 60 healthy in-
dividuals (age range: 15 to 30 years; mean age: 22
years). Out of 60 patients, 32 were females and 28
were males. The subjects were selected from the pa-
tients who reported to the outpatient section, Depart-
ment of Periodontics, Government Dental College,
Bangalore, Karnataka, India, from September 2009
to April 2010. Ethical clearance was obtained from
the institution’s Ethical Committee and written con-
sent was obtained from all the potential subjects. Cri-
teria for selection included patients who had not
undergone prophylaxis within a year of examination,
with full complement of teeth, and without any systemic
and oral disease. Calculus score of all the selected
patients was recorded by applying the calculus index
of Greene and Vermillion,22 and dental plaque scores
were recorded by using the Schick and Ash index.23
Depending on calculus index score, individuals
were divided into four groups, each of 15 patients:
Group 1, calculus index score 0 to 0.6; Group 2, cal-
culus index score 0.7 to 1.8; Group 3, calculus index
score 1.9 to 3; and Group 4, plaque group where index
varied from 0 to 3 (Schiff reagent was used as the dis-
closing solution).24
Collection of an unstimulated 5 ml of whole saliva
was made with the help of a graduate pipette and
transferred into a sterile bottle. Saliva collected in
the first few minutes was discarded. The bottles were
stoppered, labeled, and sent to the Department of
Biochemistry and Cell Biology, Indian Institute of
Sciences, Bangalore, on ice for chemical analysis.
Saliva was chemically analyzed for orthophosphate,
pyrophosphate, and pyrophosphatase enzyme.
446
Volume 82 • Number 3
Deproteination of saliva: 5 ml of saliva collected
from each of the donors, was transferred to a sterile
centrifugal test tube. A total of 3 ml of the saliva was
treated with 5 ml of 10% trichloroacetic acid and cen-
trifuged in a centrifugal machine at 3,000 rpm for 15
minutes at room temperature to remove salivary pro-
teins. The clear, supernatant saliva was analyzed for
orthophosphate, pyrophosphate, and pyrophospha-
tase enzyme.
Estimation of orthophosphate was done as per the
procedure of Flynn et al.25 by using phosphorus re-
agent. The reagents used were molybdate solution
and phosphorus reagents of the Fiske and Subbarow26
method for the determination of orthophosphate.
Standardization was done by using potassium
dihydrogen phosphate, readings were taken by using
the Klett-Summerson photoelectric colorimeter us-
ing red filter under 660 nm wavelength, and a standard
graph was drawn by plotting the readings to obtain
a linear curve. Two aliquots of 0.1-ml supernatant
saliva were pipetted out into a sterile test tube and
1 ml of the molybdate solution and 0.5 ml of the
phosphorus reagent were added, turning the color
blue; the volume was increased to 10 ml by adding
8.4 ml of distilled water. The readings were taken
and compared with a standard (a blank prepared with
1 ml of molybdate solution, 0.5 ml of phosphorus re-
agent, and 8.5 ml of distilled water) and the readings
were recorded.
The orthophosphate activity in saliva is expressed
in millimoles (mM). A total of 1 mM of orthophosphate
is equal to 136 mg of potassium dihydrogen ortho-
phosphate per milliliter.
Reading of the test reading of the blank x 1 = test blank x 1
136 standard
Estimation of pyrophosphate was done as per the
procedure of Flynn et al.25 Two aliquots of 0.1 ml
saliva were taken in a test tube and to this 0.1 ml
of 1 N HCl was added and heated for 10 minutes
at 100C for hydrolysis of the remaining pyrophos-
phate to orthophosphate. After heating, the aliquots
were cooled at room temperature and were analyzed
to give a measure of the total phosphate in the sa-
liva. The difference of the values of the phosphate
between the hydrolyzed saliva (X) and the unhydro-
lyzed saliva (Y) was taken as pyrophosphate. A blank
was prepared and the readings were recorded as was
done for orthophosphate. The following formula was
used:
Calculation: X Y = pyrophosphate
Assay of enzyme pyrophosphatase was done as fol-
lows. The chemicals prepared were veroneal acetate
buffer (0.05 M), magnesium chloride (0.1 M), and
J Periodontol • March 2011
sodium pyrophosphate (0.01 M). Standardization was
done by using clear supernatant concentrated saliva
at different volumes (0.02, 0.04, 0.08, and 1 ml)
and a standard graph was drawn and 0.04 volume
was standardized for the procedure. Test sample
was prepared as by Heppel and Hilmore.27 A total
of 1.16 ml and 1.21 ml of veroneal acetate buffer
was pipetted out to the sterile test tubes, and 0.05
ml of magnesium chloride was added. To the test tube
of veroneal acetate buffer 1.16 ml, 0.05 ml of so-
dium pyrophosphate substrate was added and later
0.04 ml of supernatant saliva was added to both the
test tubes. The assay mixture was mixed well, and af-
ter 15 minutes, reaction was arrested by adding 0.2 ml
of 10% trichloroacetic acid and the aliquot were
analyzed for orthophosphate as per the procedure
of Flynn et al.25 The difference in values between
the aliquot with the substrate and without the sub-
strate gave the presence of enzyme in saliva. A blank
was also prepared on the side that contained no
enzyme solution or saliva. One unit of enzyme pyro-
phosphatase activity was defined as 1 mM of phos-
phate released per minute.
All four groups were compared for the values of or-
thophosphate, pyrophosphate, and pyrophosphatase
by one-way analysis of variance. Scheffe test was
applied to make the intergroup comparisons.
RESULTS
Five subjects did not complete the study and were ex-
cluded from the analysis. The mean values of ortho-
phosphate, pyrophosphate, and pyrophosphatase
are given in Table1. The difference in values of ortho-
phosphate between Groups 1 and 2, Groups 1 and 3,
Groups 2 and 3, Groups 2 and 4, and Groups 3
and 4 was found to be highly significant (Table 2).
The difference in values of pyrophosphate between
Groups 1 and 3, Groups 2 and 4, and Groups 3 and
4 was found to be highly significant (Table 3). The
difference in values of pyrophosphatase between
Groups 1 and 3, Groups 2 and 3, Groups 2 and 4,
and Groups 3 and 4 was found to be highly significant
(Table 4).
Table 1.
Descriptive Statistics (mean – SD)
Group n Orthophosphate (mM)
1 13 0.2559 – 0.20774
2 14 1.3639 – 0.20831
3 14 1.7311 – 0.24497
4 14 0.1868 – 0.12409
Pradeep, Esha, Arjun Raju, Rao, Faizuddin
DISCUSSION
Calculus formation and inhibition is the result of the
interaction of certain important components present
in saliva, namely orthophosphate, pyrophosphate,
and pyrophosphatase. Enzyme alkaline phospha-
tase9 present in saliva and in plaque releases inor-
ganic orthophosphate from organic phosphate,
increasing concentration of orthophosphate locally,
which reacts with calcium ions leading to precipitation
of insoluble calcium apatite crystals. Pyrophosphate,
a byproduct of many biosynthetic reactions,10 pres-
ent in saliva inhibits crystallization and competes
with orthophosphate1 for minerals, thus having a
strong inhibitory effect on plaque mineralization.
The enzyme pyrophosphatase plays an exciting role
in formation of calculus by hydrolyzing9,10 pyrophos-
phate to orthophosphate, thus removing its inhibitory
power and simultaneously converting into ‘‘booster’’
by increasing the concentration of orthophosphate.
In the present investigation an attempt was made
to discover the role of orthophosphate, pyrophos-
phate, and pyrophosphatase in saliva in relation to
calculus formation and inhibition and also to ascer-
tain the significant relationship between them in the
calculus groups and the plaque group.
Volpe-Manhold index considers the lingual sur-
faces of mandibular six anteriors for assessing the
amount of calculus.28 We have considered here the
orthophosphate, pyrophosphate, and pyrophospha-
tase concentration of whole saliva and not just sub-
mandibular saliva. Therefore, the calculus index,22
which considers the six index teeth including the max-
illary molars (parotid duct opens opposite the crown
of the upper second molar), was taken to assess the
amount of calculus.
The pyrophosphate assay as per Flynn et al.25 was
used because it is a simple and convenient method for
the determination of pyrophosphate. It is a colorimet-
ric method that involves simple reagents easily avail-
able in the laboratory and it is also cost effective. The
method of Flynn et al.25 uses the reagents of the Fiske
and Subbarow26 method, which is considered a more
reliable method than other methods, such as that of
Pyrophosphate (mM) Pyrophosphatase (units/ml)
0.3258 – 0.12099 10.7937 – 1.95036
0.1091 – 0.10818 15.4249 – 3.15730
0.0314 – 0.03319 27.2900 – 4.22250
0.3860 – 0.23809 7.5427 – 2.35186
447
Orthophosphate, Pyrophosphate, and Pyrophosphatase in Saliva Volume 82 • Number 3

Table 2. 1.7311 mM, which is significantly different from the

values in Groups 1 and 2. The mean value of ortho-
Scheffé Test (orthophosphate)
phosphate in Group 4 is 0.1868 mM. It is observed
that the mean of orthophosphate declined consider-
Groups Mean Difference P Value
ably in Group 4, the plaque group, compared to
1 and 2 -1.1079533 <0.001* Groups 1, 2, and 3, the calculus groups. The observa-
tion is in agreement with the observations of Sawinski
1 and 3 -1.4751867 <0.001*
and Cole11 that the severity of the calculus formation
1 and 4 0.0690800 0.910 is directly proportional to the orthophosphate content.
Also, in the study by Sawinski and Cole,11 the values
2 and 3 -0.3672333 <0.001*
are calculated in mg%. The mean value of orthophos-
2 and 4 1.1770333 <0.001* phate was found to be 7.36 mg%, which is equal to
0.782 mM, which is lower than our value (1.7311
3 and 4 1.5442667 <0.001*
mM = 15.98 mg%).
* P <0.05 significant. Further, the values of orthophosphate in our study
are considerably lower than those found by Vogel
et al.8 There may be various reasons which might
Table 3. have contributed to these lower values. It has been
Scheffé Test (pyrophosphate) suggested that after eating carbohydrates (which
lowers plasma phosphate), the phosphate concen-
tration of saliva decreases.30 This may be the reason
Groups Mean Difference P Value
for lower values of phosphate concentration in an In-
1 and 2 0.216700 0.011 dian population, as consumption of foods rich in car-
bohydrates are common in Indians, especially in
1 and 3 0.294440 <0.001*
the south Indian population. Daily rhythms in secre-
1 and 4 -0.060207 0.894 tion rate and composition of saliva have been studied,
and it has been found that minimum amplitude for
2 and 3 0.077740 0.767
phosphate concentration is at 4:00 am.30 In our study,
2 and 4 -0.276907 <0.001* samples were collected at about 7:00 am, which can
be another reason for the lower values of phosphate
3 and 4 -0.354647 <0.001*
concentration. It is a proven fact that with increased
* P <0.05 significant.
flow, phosphate concentration in saliva decreases.30
So, the increased flow may also have caused lower
values of phosphate concentration in our study. To
Table 4.
our knowledge, our study is the first to assess the
Scheffé Test (pyrophosphatase) levels of salivary orthophosphate and pyrophosphate
concentration in an Indian population. Further studies
Groups Mean Difference P Value are needed to assess the salivary inorganic phos-
phate concentrations in subjects, taking into con-
1 and 2 -4.63127 0.04
sideration the various factors that might affect the
1 and 3 -16.49633 <0.001* salivary inorganic phosphate concentration like
carbohydrate-rich diet, circadian rhythm, and flow
1 and 4 3.25093 0.092
rate of saliva.
2 and 3 -11.86507 <0.001* The mean pyrophosphate value in Group 1 is
0.3258 mM, whereas in Group 2 it is 0.1091 mM,
2 and 4 7.88220 <0.001*
the difference not being statistically significant. The
3 and 4 19.74727 <0.001* mean pyrophosphate value in Group 3 is 0.0314
* P <0.05 significant. mM, and the difference between Groups 1 and 3 is
statistically significant. In Group 4 the mean value
Kuttner and Lichtenstien (in Varley29). The duplica- of pyrophosphate is 0.3860 mM, and the difference
tion of samples was done to eliminate error. between Groups 3 and 4 and Groups 2 and 4 is sta-
The results indicate that in Group 1 mean level of tistically significant. Hence, pyrophosphate con-
orthophosphate is 0.2559 mM and in Group 2 it is centration is inversely proportional to the extent of
1.3639 mM, which is about four times higher than calculus formation (i.e., as the concentration of pyro-
Group 1; this difference is statistically significant. Sim- phosphate increases, the calculus score shows a steady
ilarly, the mean level of orthophosphate in Group 3 is decrease). This is highly suggestive of an inhibitory

448
J Periodontol • March 2011
role of pyrophosphate in the formation of calculus
and also concurs with the previous observation made
by Sawinski and Cole.11 Similar observations in relation
to pyrophosphate have been reported by Vogel and
Amdur, 8 Bisaz et al., 21 and Edgar and Jenkins12
in parotid and submaxillary saliva, except for the fact
that there is a variation in concentration of pyrophos-
phate in whole saliva than the other two sources.
Also included in the investigation is the assay of
enzyme pyrophosphatase, which was carried out in
the whole saliva to discover the relationship between
calculus formation and inhibition. The mean level
of pyrophosphatase in Group 1 is 10.7937 units/ml
and in Group 2 it is 15.4249 units/ml. The difference
between the mean values of Groups 1 and 2 is not
statistically significant. The mean value of the pyro-
phosphatase activity in Group 3 is 27.2900 units/ml.
The difference in the pyrophosphatase values in
Groups 1 and 3 and Groups 2 and 3 is statistically sig-
nificant. The mean value of the pyrophosphatase ac-
tivity in Group 4 is 7.5427 units/ml and its difference
with Groups 2 and 3 is statistically significant. This
shows that the level of pyrophosphatase increases
with the calculus score, thus playing a role in the for-
mation of calculus.
Thus, the results are conclusive that the compo-
nents orthophosphate, pyrophosphate, and pyrophos-
phatase present in saliva have a very significant role
to play in formation and inhibition of calculus. This
study again reinforces the idea of using pyrophosphate
as an anticalculus agent. Pyrophosphate was first
tested as an anticalculus agent by Kinoshita and
Mühlemann,31 who investigated a troche containing
2.9% pyrophosphate using the 7-day mylar foil
model. A decrease in calculus formation of 14.6%
was observed in the test group, but this difference
was not statistically significant. The use of pyrophos-
phate as a potential anticalculus agent was sus-
pended until 1985, possibly as a result of these
findings. Since then there have been various clinical
trials that have used pyrophosphate-based combi-
nations with sodium fluoride or copolymer as poten-
tial anticalculus agents.32-37
Bisphosphonates represent another group of syn-
thetic pyrophosphate analogs that interact strongly
with minerals38 and are thought to prevent calculus
deposition by inhibiting crystal growth, and have been
used as potential anticalculus agents. Mühlemann
et al.39 investigated the efficacy of ethane-1-hydroxy-
1,1-diphosphonate (EHDP) on the accumulation of
calculus in known calculus formers and found that
the EHDP solution reduced calculus formation by
49% and by 16% in rapid and slow calculus formers,
respectively. Herforth40 tested the use of a sodium
etidronate mouthrinse using the mylar foil model
and found that solutions containing 1% and 0.1%
Pradeep, Esha, Arjun Raju, Rao, Faizuddin
EHDP reduced calculus formation by 56.2% and
by 56.8%, respectively. Inhibitory effects of a novel
bisphosphonate, TRK-530 (disodium dihydrogen
[4-(methylthio)phenylthio] methanebisphosphonate),
were studied on dental calculus formation in rats
and it was found that TRK-530 inhibited the forma-
tion of dental calculus in a dose-dependent fashion
via a local effect by inhibition of the precipitation of
calcium-phosphate from solution.41
A number of anticalculus or antitartar dentifrices
containing pyrophosphates and bisphosphonates
have been developed and proved to be efficacious.
Sturzenberger et al.42 studied the effects of a 3% EHDP
dentifrice on 64 subjects who were prescreened for
calculus-forming potential and then received a dental
prophylaxis. At the conclusion of the study, the sub-
jects who had used the sodium etidronate dentifrice
were divided equally. One subgroup continued to
use the sodium etidronate dentifrice, whereas the
other used placebo. The purpose was to determine
whether the test dentifrice could remove a quantity
of preformed calculus over 8 weeks. Sodium etidro-
nate dentifrice reduced preformed calculus levels by
25% with no adverse reactions. Suomi et al.43 carried
out an 18-month clinical trial to compare the effect
of a 3% sodium etidronate/0.22% sodium fluoride
dentifrice to a control dentifrice. Levels of supragingi-
val calculus were reduced by 27.1% after 6 months, by
39.9% after 12 months, and by 42.1% after 18 months
compared to controls. Gaffar and Moreno44 tested
the in vitro and in vivo effect of a monophosphonate
2-phosphonobutane 1,2,4, tricarboxylate (PBTA) on
crystal growth. The in vivo study, in which a 1% PBTA
solution was applied topically to the teeth of rats,
demonstrated a significant decrease in the incidence
of calculus formation. Recently, the anticalculus ef-
fect of a triclosan mouthwash containing phytate
(inositol hexaphosphate) was tested in a double-
masked, randomized, three-period crossover trial.
Phytate was found to be an effective anticalculus
agent.45 Hence, further long-term prospective stud-
ies are needed to confirm these findings.
In 2000, Ho et al.46 reported work on the gene for
progressive joint ankylosis in mice, known as ANK.
Recent data showed that human analog of this ANKH
gene may play a role in familial calcium pyrophos-
phate dihydrate deposition (CPPD) disease, which
involves the deposition of CPPD microcrystals in the
joint fluid, cartilage, and periarticular tissues.47 This
gene encodes a protein expressed on cell mem-
branes.47 Cell transfection experiments established
that the protein carried pyrophosphate ions from the
intracellular to the extracellular compartment. Pro-
benecid, which was previously shown to decrease
the amount of pyrophosphate released by cultured
chondrocytes,48 was found to inhibit transport by
449
Orthophosphate, Pyrophosphate, and Pyrophosphatase in Saliva
the ANK protein. The mutation in ank/ank mice
therefore results in a decrease in extracellular pyro-
phosphate levels resulting in apatite deposit within
tissues. In humans, conversely, extracellular pyro-
phosphate levels increase.47 The elevation in joint
fluid pyrophosphate levels promotes the formation
of CPPD crystals within joint tissues. Pyrophosphate
plays a dual role in the pathophysiology of the joint
lesions, inhibiting the formation of apatite deposits
and promoting the deposition of CPPD. These data
explain how different mutations affecting the same
gene can lead to the formation of different crystal
types.49 Further studies should be carried out to
relate the dentition and dental calculus index in pa-
tients with proven ANKH mutations and to determine
if the mutation in this gene plays any important role
in increased dental calculus formation.
CONCLUSIONS
Until the mid-20th century, calculus was the king of
the etiologic hierarchy in periodontal disease. In the
past 25 years, however, calculus has been deposed
by plaque, and the hardened criminal has come to
be viewed as a fossilized remnant of minor signifi-
cance. This shift in perception became most apparent
in the 1960s, and was largely a response to the find-
ings that supragingival and subgingival calculus was
mineralized plaque covered by an unmineralized
bacterial layer. Initial damage to the gingival margin
is presumably the result of enzymatic effects caused
by the microorganisms of plaque, but this process is
enhanced by the formation of supragingival dental
calculus, which provides further retention and thus
promotes new plaque accumulations. A holistic ap-
proach toward the control of periodontal disease
should not only include antiplaque agents but also
anticalculus agents. This emphasizes the importance
of regular removal of calcified deposits in periodontal
therapy and of the clinical value of anticalculus den-
tifrices. This study concludes that the components or-
thophosphate, pyrophosphate, and pyrophosphatase
present in saliva have a very significant role to play in
formation and inhibition of calculus. Pyrophosphate-
containing dentifrices are potential anticalculus
agents; therefore further studies should be carried
out to assess the anticalculus efficacy of newer bi-
sphosphonates.
ACKNOWLEDGMENTS
The authors thank the staff at the Department of Bio-
chemistry, Indian Institute of Science, Bangalore, for
guiding and providing all the facilities to carry out
the biochemical investigations, and Mr. Jagannatha,
the statistician, for his valuable guidance. The authors
report no conflicts of interest related to this study.
450
Volume 82 • Number 3
REFERENCES
1. White DJ. Dental calculus: Recent insights into occur-
rence, formation, prevention, removal and oral health
effects of supragingival and subgingival deposits. Eur
J Oral Sci 1997;105:508-522.
2. Greene JC. Oral hygiene and periodontal disease. Am
J Public Health Nations Health 1963;53:913-922.
3. Russell AL. International nutrition surveys: A summary
of preliminary dental findings. J Dent Res 1963;42:
233-244.
4. Friskopp J, Hammarström L. A comparative, scan-
ning electron microscopic study of supragingival and
subgingival calculus. J Periodontol 1980;51:553-562.
5. Patters MR, Landesberg RL, Johansson LA, Trummel
CL, Robertson PB. Bacteroides gingivalis antigens and
bone resorbing activity in root surface fractions of
periodontally involved teeth. J Periodontal Res 1982;
17:122-130.
6. Mandel ID, Gaffar A. Calculus revisited. A review. J
Clin Periodontol 1986;13:249-257.
7. Modéer T, Wondimu B. Periodontal diseases in chil-
dren and adolescents. Dent Clin North Am 2000;44:
633-658.
8. Vogel JJ, Amdur BH. Inorganic pyrophosphate in
parotid saliva and its relation to calculus formation.
Arch Oral Biol 1967;12:159-163.
9. Jenkins GN. Pellicle, plaque and calculus. In: The
Physiology and Biochemistry of the Mouth, 4th edi-
tion. Oxford: Blackwell Scientific Publications; 1978:
122-123.
10. Alcock NW, Shils ME. Association of inorganic pyro-
phosphatase activity with normal calcification of rat
costal cartilage in vivo. J Biochem 1969;12:505-510.
11. Sawinski VJ, Cole DF. Phosphate concentration of
sterile human parotid saliva and its relationship to
dental disorders. J Dent Res 1965;44:827.
12. Edgar WM, Jenkins GN. Inorganic pyrophosphate in
human parotid saliva and dental plaque. Arch Oral
Biol 1972;17:219-223.
13. Hausmann E, Bisaz S, Russel RG, Fleisch H. The
concentration of inorganic pyrophosphate in human
saliva and dental calculus. Arch Oral Biol 1970;15:
1389-1392.
14. Draus FJ, Lesniewski M, Miklos FL. Pyrophosphate
and hexametaphosphate effects in in vitro calculus
formation. Arch Oral Biol 1970;15:893-896.
15. Fleisch H, Bisaz S, Care AD. Effect of orthophosphate
on urinary pyrophosphate excretion and the preven-
tion of urolithiasis. Lancet 1964;201:1065-1067.
16. Fleisch H, Bisaz S. Isolation from urine of pyrophos-
phate, a calcification inhibitor. Am J Physiol 1962;203:
671-675.
17. Fleisch H, Russell RG, Straumann F. Effects of pyro-
phosphate on hydroxyapatite and its implication in
calcium homeostasis. Nature 1966;212:901-903.
18. Fleisch H, Neuman WF. Mechanisms of calcification:
Role of collagen, polyphosphates and phosphatase.
Am J Physiol 1961;200:1296-1300.
19. Perkins HR, Walker PG. The occurrence of pyrophos-
phate in bone. J Bone Joint Surg Br 1958;40-B:333-339.
20. Thomas WC, Howard JE. Studies on the mineralizing
propensity of urine from patients with and without renal
calculi. Trans Assoc Am Physicians 1959;72:181-187.
21. Bisaz S, Russell RG, Fleisch H. Isolation of inorganic
pyrophosphate from bovine and human teeth. Arch
Oral Biol 1968;13:683-696.
J Periodontol • March 2011
22. Greene JC, Vermillion JR. The simplified oral hygiene
index. J Am Dent Assoc 1964;68:7-13.
23. Schick RA, Ash MM. Evaluation of vertical method of
tooth brushing. J Periodontol 1961;32:346-353.
24. Arnim SS. The use of disclosing agents for measuring
tooth cleanliness. J Periodontol 1963;34:227-243.
25. Flynn RM, Jones ME, Lipmann F. A colorimetric deter-
mination of inorganic pyrophosphate. J Biol Chem 1954;
211:791-796.
26. Fiske CH, Subbarow YJ. The coloro- metric determi-
nation of phosphorus. J Biol Chem 1925;66:375-400.
27. Heppel LA, Hilmore RJ. Purification of yeast inorganic
pyrophosphatase. J Biol Chem 1951;192:87-94.
28. Volpe AR, Manhold JH, Hazen SP. In vivo assessment -
Part I – A method and its examiners reproducibility. J
Periodontol 1965;36:292-298.
29. Varley H. Calcium, magnesium and phosphate. In:
Practical Clinical Biochemistry, 4th edition. New Delhi:
CBS Publishers; 1967:446-448.
30. Jenkins GN. Saliva. The Physiology and Biochemistry
of the Mouth, 4th ed. Oxford: Blackwell Scientific
Publications; 1978:304-315.
31. Kinoshita S, Mühlemann HR. Effect of sodium ortho-
and pyrophosphate on supragingival calculus. Helv
Odontol Acta 1966;10:46-48.
32. Lobene RR. A study to compare the effects of two
dentifrices on adult dental calculus formation. J Clin
Dent 1989;1:67-69.
33. Taner IL, Kebudi E, Taplamacioğlu B. A clinical study
to evaluate the anticalculus effect of a dentifrice on
calculus formation [in Turkish]. Ankara Univ Hekim
Fak Derg 1990;17:45-49.
34. Gaengler P, Kurbad A, Weinert W. Evaluation of anti-
calculus efficacy. An SEM method of evaluating the
effectiveness of pyrophosphate dentifrice on calculus
formation. J Clin Periodontol 1993;20:144-146.
35. Banoczy J, Sari K, Schiff T, Petrone M, Davies R, Volpe
AR. Anticalculus efficacy of three dentifrices. Am J
Dent 1995;8:205-208.
36. Triratana T, Kraivaphan P, Tandhachoon K, Rustogi K,
Volpe AR, Petrone M. Effect of a pre-brush mouthrinse
containing triclosan and a copolymer on calculus for-
mation: A three-month clinical study in Thailand. J Clin
Dent 1995;6:139-141.
37. Fairbrother KJ, Kowolik MJ, Curzon MEJ, et al. The
comparative clinical efficacy of pyrophosphate/triclosan,
copolymer/triclosan and zinc citrate/triclosan dentifrices
for the reduction of supragingival calculus formation. J
Clin Dent 1997;8(Spec. No. 2):62-66.
38. Fleisch H, Russell RG. A review of the physiological
and pharmacological effects of pyrophosphate and
Pradeep, Esha, Arjun Raju, Rao, Faizuddin
diphosphonates on bones and teeth. J Dent Res 1972;
51:324-332.
39. Mühlemann HR, Bowles D, Schait A, Bernimoulin JP.
Effect of diphosphonate on human supragingival cal-
culus. Helv Odontol Acta 1970;14:31-33.
40. Herforth VA. Clinical investigations about the tartar
restraining effects of HEDP [in German]. Deutsche
Zahnartzl 1976;31:392-395.
41. Sikder MN, Itoh M, Iwatsuki N, Shinoda H. Inhibitory
effect of a novel bisphosphonate, TRK-530, on dental
calculus formation in rats. J Periodontol 2004;75:537-
545.
42. Sturzenberger OP, Swancar JR, Reiter G. Reduction of
dental calculus in humans through the use of a denti-
frice containing a crystal-growth inhibitor. J Periodon-
tol 1971;42:416-419.
43. Suomi JD, Horowitz HS, Barbano JP, Spolsky VW,
Heifetz SB. A clinical trial of a calculus-inhibitory
dentifrice. J Periodontol 1974;45:139-145.
44. Gaffar A, Moreno EC. Evaluation of 2-phosphono-
butane 1,2,4 tricarboxylate as a crystal growth
inhibitor in vitro and in vivo. J Dent Res 1985;64:
6-11.
45. Grases F, Perelló J, Sanchis P, et al. Anticalculus effect
of a triclosan mouthwash containing phytate: A double-
blind, randomized, three-period crossover trial. J Peri-
odontal Res 2009;44:616-621.
46. Ho AM, Johnson MD, Kingsley DM. Role of the mouse
ank gene in control of tissue calcification and arthritis.
Science 2000;289:265-270.
47. Pendleton A, Johnson MD, Hughes A, et al. Mutations in
ANKH cause chondrocalcinosis due to calcium pyro-
phosphate crystal deposition. Am J Hum Genet 2002;
71:933-940.
48. Rosenthal AK, Ryan LM. Probenecid inhibits trans-
forming growth factor-b 1 induced pyrophosphate
elaboration by chondrocytes. J Rheumatol 1994;21:
896-900.
49. Reichenberger E, Tiziani V, Watanabe S, et al. Autoso-
mal dominant craniometaphyseal dysplasia is caused
by mutations in the transmembrane protein ANK. Am J
Hum Genet 2001;68:1321-1326.
Correspondence: Professor Avani R. Pradeep, Department
of Periodontics, Government Dental College and Research
Institute, Fort Bangalore-560002, Karnataka, India. Fax: 91-
80-26703176; e-mail: periodontics_gdc@rediffmail.com.
Submitted June 10, 2010; accepted for publication August
5, 2010.
451